DESCRIPTION: This project addresses the molecular mechanisms underlying signal transduction and regulation of muscarinic cholinergic receptors. During the previous project period, the m1, m2, and m3 receptors were modified by site-directed mutagenesis to define domains relevant to second messenger signaling, internalization, and downregulation. The present proposal focuses on the direct interactions of the muscarinic receptors with proteins involved in receptor functions. These include a) coupling to specific G proteins, b) membrane-delimited interactions with ion channels, c) phosphorylation by protein kinases, and d) aggregation among the receptors themselves and with other membrane proteins. Aggregation of integral membrane proteins is thermodynamically favored, and G protein coupled receptors and ion channels tends to form homo-oligomers (as reported for the m2 receptor) or hetero-oligomers. Hetero-aggregation has not been adequately investigated for G protein-coupled receptors; yet, aggregates may play essential roles in all receptor functions. Methodologies will be developed to study receptor aggregation, using biophysical, biochemical, aand genetic approaches in order to understand receptor function at the quaternary structural level of membrane protein organization. This study should also address the question as to whether muscarinic receptors can assume multiple conformations, each signalling along distinct pathways. The expected insights from the studies outlined in this proposal should assist in the design of cholinergic therapy for cognitive disorders.